This is an R01 application in response to FOA PAR-12-060 Solicitation of Validated Hits for the Discovery of in vivo Chemical Probe (R01). cAMP-mediated cell signaling regulates a myriad of important biological processes and plays important roles in the development of many human diseases. In eukaryotic cells, the effects of cAMP are mainly transduced by two groups of intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and a new family of more recently discovered exchange proteins directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPACs/cAMP-GEFs). One of the major challenges within the research field is the lack of EPAC specific antagonists for interrogating the biological functions of EPACs in physiological setting and for understanding of disease mechanisms in which EPACs are implicated. To bridge this gap, we have identified and validated several novel, first-in-class small chemical antagonists specific for EPACs. In the present proposal, we will build on the validated hits discovered in our laboratory as the lead candidates and design, synthesize and evaluate more potent and specific EPAC specific probes. Optimized EPAC chemical probes that effectively inhibit EPAC signaling in biochemical and cell-based assays will be analyzed in vivo in animal models to identify potential target molecules for development of EPAC-based therapeutics. The proposed studies build upon an ongoing, proven productive collaboration between two principal investigators with extensive complimentary expertise in EPAC/cAMP biology and medicinal chemistry, respectively. The interdisciplinary perspectives, new chemical entities and emerging medical importance of EPAC will lead to the discovery of a new class of in vivo chemical probes that can be used in studying disease mechanisms and treatments related to EPAC signaling.